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  • 99
    New England Biolabs desthiobiotin gtp
    Desthiobiotin Gtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gtp
    The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting <t>TEC</t> ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM <t>GTP</t> in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.
    Gtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gtp
    IMPDH-based cytoophidia in iPSCs respond to <t>GTP</t> levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and <t>dCTP</t> was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments
    Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1073 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore guanosine triphosphate gtp
    (A) Time-dependent release of DOX-D from the <t>ATP-responsive</t> hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM <t>GTP,</t> and (d) untreated.
    Guanosine Triphosphate Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher guanosine triphosphate gtp
    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active <t>(Rac1-GTP)</t> and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p
    Guanosine Triphosphate Gtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TriLink guanosine triphosphate gtp
    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active <t>(Rac1-GTP)</t> and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p
    Guanosine Triphosphate Gtp, supplied by TriLink, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim guanosine triphosphate gtp
    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active <t>(Rac1-GTP)</t> and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p
    Guanosine Triphosphate Gtp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Valiant guanosine triphosphate gtp
    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active <t>(Rac1-GTP)</t> and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p
    Guanosine Triphosphate Gtp, supplied by Valiant, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Fisher Scientific guanosine triphosphate gtp
    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active <t>(Rac1-GTP)</t> and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p
    Guanosine Triphosphate Gtp, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche guanosine triphosphate gtp
    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both <t>ATP</t> and <t>GTP</t> (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P
    Guanosine Triphosphate Gtp, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cytoskeleton Inc guanosine triphosphate gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer guanosine triphosphate gtp biotin 11 gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp Biotin 11 Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cytoskeleton Inc rho guanosine triphosphate gtp pulldown assay kit
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Rho Guanosine Triphosphate Gtp Pulldown Assay Kit, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore guanosine triphosphate sodium salt gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Sodium Salt Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    GE Healthcare guanosine triphosphate gtp binding protein
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp Binding Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 81/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cytoskeleton Inc guanosine triphosphate gamma s gtpγs
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gamma S Gtpγs, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    List Biological Laboratories guanosine triphosphate gtp binding protein
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp Binding Protein, supplied by List Biological Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher n methylanthraniloyl guanosine triphosphate
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    N Methylanthraniloyl Guanosine Triphosphate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Moravek Biochemicals c gtp
    Effect of Archease on the single-turnover rate of RNA ligation by RtcB. ( A ) RNA ligation reactions with RtcB alone or with the inclusion of 100 nM Archease. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), <t>NaCl</t> (300 mM), MnCl 2 (0.25 mM), <t>GTP</t> (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. ( B ) Plots of ligation product formation over time fitted to a single-exponential equation. Values are the mean ± SE for two separate experiments.
    C Gtp, supplied by Moravek Biochemicals, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jena Bioscience gtp
    Effect of Archease on the single-turnover rate of RNA ligation by RtcB. ( A ) RNA ligation reactions with RtcB alone or with the inclusion of 100 nM Archease. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), <t>NaCl</t> (300 mM), MnCl 2 (0.25 mM), <t>GTP</t> (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. ( B ) Plots of ligation product formation over time fitted to a single-exponential equation. Values are the mean ± SE for two separate experiments.
    Gtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jena Bioscience mant gtp
    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than <t>GTP/GDP</t> interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
    Mant Gtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gtp  (TaKaRa)
    91
    TaKaRa gtp
    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than <t>GTP/GDP</t> interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
    Gtp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rap1 guanosine triphosphate gtp
    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than <t>GTP/GDP</t> interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
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    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than <t>GTP/GDP</t> interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
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    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than <t>GTP/GDP</t> interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background
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    Phosphorylation of the XJ TGB1 protein in vitro and in vivo . (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJ TGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJ TGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of <t>[γ-</t> 32 P]ATP or [γ- 32 <t>P]GTP.</t> After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJ TGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJ TGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJ TGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJ RNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJ TGB1 protein. (D) In vivo phosphorylation of XJ TGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C . (This figure is available in colour at JXB online.)
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    Bleomycin decreased vascular endothelial growth factor receptor 2 (VEGFR2) and guanosine triphosphate <t>cyclohydrolase-1</t> <t>(GTPCH-1)</t> levels in WT mice. VEGFR2, endothelial nitric oxide synthase (eNOS) and GTPCH-1 protein content in the lung were evaluated by western blot analysis in WT and R 213 G mice at 22 days of age after exposure to PBS (10 μL for 9 doses) or Bleomycin (3 units/kg/dose dissolved in 10 μL of PBS for 9 doses). 25 μg lung protein was loaded onto the gels. Representative blots are shown along with optical density normalized to β-actin and expressed relative to WT control mice. ( a ) VEGFR2 ( b ) eNOS ( c ) Representative blots for VEGFR2 and eNOS ( d ) GTPCH-1, ( e ) Representative blot for GTPCH-1 * p
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    Bleomycin decreased vascular endothelial growth factor receptor 2 (VEGFR2) and guanosine triphosphate <t>cyclohydrolase-1</t> <t>(GTPCH-1)</t> levels in WT mice. VEGFR2, endothelial nitric oxide synthase (eNOS) and GTPCH-1 protein content in the lung were evaluated by western blot analysis in WT and R 213 G mice at 22 days of age after exposure to PBS (10 μL for 9 doses) or Bleomycin (3 units/kg/dose dissolved in 10 μL of PBS for 9 doses). 25 μg lung protein was loaded onto the gels. Representative blots are shown along with optical density normalized to β-actin and expressed relative to WT control mice. ( a ) VEGFR2 ( b ) eNOS ( c ) Representative blots for VEGFR2 and eNOS ( d ) GTPCH-1, ( e ) Representative blot for GTPCH-1 * p
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    Bleomycin decreased vascular endothelial growth factor receptor 2 (VEGFR2) and guanosine triphosphate <t>cyclohydrolase-1</t> <t>(GTPCH-1)</t> levels in WT mice. VEGFR2, endothelial nitric oxide synthase (eNOS) and GTPCH-1 protein content in the lung were evaluated by western blot analysis in WT and R 213 G mice at 22 days of age after exposure to PBS (10 μL for 9 doses) or Bleomycin (3 units/kg/dose dissolved in 10 μL of PBS for 9 doses). 25 μg lung protein was loaded onto the gels. Representative blots are shown along with optical density normalized to β-actin and expressed relative to WT control mice. ( a ) VEGFR2 ( b ) eNOS ( c ) Representative blots for VEGFR2 and eNOS ( d ) GTPCH-1, ( e ) Representative blot for GTPCH-1 * p
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    Image Search Results


    The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting TEC ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM GTP in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting TEC ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM GTP in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Flow Cytometry

    Eco RNAP, Sce RNAPII and Hsa MT RNAP efficiently incorporate OZM in place of uridine. ( A ) The nucleic acid scaffolds employed in the experiments in (B) and (C). ( B ) Incorporation of OZM in place of uridine: the initial TECs (lanes 1 and 4; top schematics in (A)) were supplemented with UTP and GTP (lanes 2 and 3), OZM triphosphate and GTP (lanes 5 and 6), ΨTP and GTP (lanes 7 and 8). NTPs were added at 20 μM and the reactions were incubated for 2 min at 25°C. RNAs were resolved on 25% urea PAGE. Fractional misincorporations (additional bands) are evident in several lanes. ( C ) OZM triphosphate allows efficient transcription of the sequence position encoding uridine: the initial TEC (lane 1, bottom schematics in (A)) was supplemented with ATP, CTP and GTP (lane 2) and additionally with UTP (lane 3) or OZM triphosphate (lane 4). NTPs were added at 100 μM and the reactions were incubated for 15 s at 25°C. The limited read through the position encoding uridine in the absence of UTP and OZM triphosphate (lane 2) was likely due to the misincorporation of CMP in place of UMP. Each assay was performed in triplicate. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel.

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: Eco RNAP, Sce RNAPII and Hsa MT RNAP efficiently incorporate OZM in place of uridine. ( A ) The nucleic acid scaffolds employed in the experiments in (B) and (C). ( B ) Incorporation of OZM in place of uridine: the initial TECs (lanes 1 and 4; top schematics in (A)) were supplemented with UTP and GTP (lanes 2 and 3), OZM triphosphate and GTP (lanes 5 and 6), ΨTP and GTP (lanes 7 and 8). NTPs were added at 20 μM and the reactions were incubated for 2 min at 25°C. RNAs were resolved on 25% urea PAGE. Fractional misincorporations (additional bands) are evident in several lanes. ( C ) OZM triphosphate allows efficient transcription of the sequence position encoding uridine: the initial TEC (lane 1, bottom schematics in (A)) was supplemented with ATP, CTP and GTP (lane 2) and additionally with UTP (lane 3) or OZM triphosphate (lane 4). NTPs were added at 100 μM and the reactions were incubated for 15 s at 25°C. The limited read through the position encoding uridine in the absence of UTP and OZM triphosphate (lane 2) was likely due to the misincorporation of CMP in place of UMP. Each assay was performed in triplicate. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel.

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Sequencing

    The effect of OZM on transcription through polythymidine sequences by Eco RNAP. ( A ) TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( B ) Lane profiles from gels in (A) depicting the effect of GreA on the transcription of four- and seven-thymidine tracts. ( C ) TECs arrested at the four-thymidine tract (S326 template) were purified by gel-filtration and supplemented with 100 μM NTPs in the absence and presence of 2μM GreA or GreB for 5 min at 25°C.

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: The effect of OZM on transcription through polythymidine sequences by Eco RNAP. ( A ) TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( B ) Lane profiles from gels in (A) depicting the effect of GreA on the transcription of four- and seven-thymidine tracts. ( C ) TECs arrested at the four-thymidine tract (S326 template) were purified by gel-filtration and supplemented with 100 μM NTPs in the absence and presence of 2μM GreA or GreB for 5 min at 25°C.

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Purification, Filtration

    The effect of OZM on processive transcription by Sce RNAPII and Hsa MT RNAP. TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( A ) Transcription through the four-thymidine tract. ( B ) Transcription through the OZM-responsive arrest site. OZM lane traces are shown to the right, the Eco RNAP trace was quantified from the gel in Figure 6B .

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: The effect of OZM on processive transcription by Sce RNAPII and Hsa MT RNAP. TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( A ) Transcription through the four-thymidine tract. ( B ) Transcription through the OZM-responsive arrest site. OZM lane traces are shown to the right, the Eco RNAP trace was quantified from the gel in Figure 6B .

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques:

    Transcription through a template encoding an OZM-responsive arrest site by Eco RNAP. TECs were assembled using the scaffolds shown to the right of the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM-triphosphate for 5 min at 25°C in the presence or absence of 2 μM GreA. The sequence corresponding to the annealing region of the RNA primer is underlined, thymidines in the transcribed region are highlighted. Each assay was performed in triplicate. ( A ) Transcription through two representative short templates. The lane profiles depict the effect of OZM on transcription of the S245 template (left gel panel). Pixel counts were linearly scaled to span 256 gradations within each gel panel. Gels were pseudocolored using the RGB lookup table shown to the right of the gel panels to visualize the low intensity bands. ( B ) Transcription through the simplified and longer derivative of the S275 template (right gel in A) that encodes the OZM-responsive arrest site. The OZM-responsive arrest site at +7 encodes the main determinants of a consensus pause: pyrimidine at the RNA 3′ end, purine at +1 and Gs at −11 and −10, these determinants are shown in bold. Pixel counts were linearly scaled to span the full 8-bit grayscale range. Lane profiles quantified from the gel are presented on the right. The RNA rescued from the arrest site by GreA in OZM-chase does not quantitatively reappear upstream or downstream of the arrest site (see the text for details and possible explanations).

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: Transcription through a template encoding an OZM-responsive arrest site by Eco RNAP. TECs were assembled using the scaffolds shown to the right of the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM-triphosphate for 5 min at 25°C in the presence or absence of 2 μM GreA. The sequence corresponding to the annealing region of the RNA primer is underlined, thymidines in the transcribed region are highlighted. Each assay was performed in triplicate. ( A ) Transcription through two representative short templates. The lane profiles depict the effect of OZM on transcription of the S245 template (left gel panel). Pixel counts were linearly scaled to span 256 gradations within each gel panel. Gels were pseudocolored using the RGB lookup table shown to the right of the gel panels to visualize the low intensity bands. ( B ) Transcription through the simplified and longer derivative of the S275 template (right gel in A) that encodes the OZM-responsive arrest site. The OZM-responsive arrest site at +7 encodes the main determinants of a consensus pause: pyrimidine at the RNA 3′ end, purine at +1 and Gs at −11 and −10, these determinants are shown in bold. Pixel counts were linearly scaled to span the full 8-bit grayscale range. Lane profiles quantified from the gel are presented on the right. The RNA rescued from the arrest site by GreA in OZM-chase does not quantitatively reappear upstream or downstream of the arrest site (see the text for details and possible explanations).

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Sequencing

    IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments

    Journal: Cell Division

    Article Title: IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

    doi: 10.1186/s13008-018-0038-0

    Figure Lengend Snippet: IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments

    Article Snippet: DON (Sigma #D2141), ribavirin (Abcam #ab120660), dCTP (Invitrogen #10217016) and GTP (Sigma #G8877) were dissolved in water.

    Techniques:

    (A) Time-dependent release of DOX-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM GTP, and (d) untreated.

    Journal: Chemical Science

    Article Title: pH- and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules †Electronic supplementary information (ESI) available: Nucleic acid sequences; characterization of nucleic acid-modified copolymers; SEM images, confocal images of microcapsules; calibration curves of TMR-D, TR-D, and DOX-D; synthesis of DOX-D; CD spectra of pH-responsive hydrogel; characterization of hydrogel membranes. See DOI: 10.1039/c6sc04770jClick here for additional data file.

    doi: 10.1039/c6sc04770j

    Figure Lengend Snippet: (A) Time-dependent release of DOX-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM GTP, and (d) untreated.

    Article Snippet: Reagents and materials Magnesium chloride, sodium chloride, doxorubicin hydrochloride (DOX), 4-carboxyphenylboronic acid, dextran (MW = 40 kDa), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES base), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES acid), N -(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDC), N -hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (MW = 30, 450, and 1250 kDa), 2-(N -morpholino)ethanesulfonic acid (MES), ammonium persulfate (APS), N ,N ,N ′,N ′-tetramethylethylenediamine (TEMED), acrylamide solution (40%), poly(allylamine hydrochloride) (PAH, MW = 58 kDa), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP) were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Derivative Assay

    (A) Time-dependent release of TMR-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). (B) Fluorescence spectra corresponding to the release of TMR-D upon exposing the microcapsules loaded with TMR-D to various concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50 and (f) 75 mM. Inset: derived calibration curve corresponding to the fluorescence intensities of the released TMR-D at λ em = 582 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of TMR-D upon the treatment of the ATP-responsive microcapsules with different nucleotide triphosphates for a fixed time interval of 30 min: (a) 25 mM ATP, (b) 25 mM TTP, (c) 25 mM CTP, (d) 25 mM GTP, and (e) untreated.

    Journal: Chemical Science

    Article Title: pH- and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules †Electronic supplementary information (ESI) available: Nucleic acid sequences; characterization of nucleic acid-modified copolymers; SEM images, confocal images of microcapsules; calibration curves of TMR-D, TR-D, and DOX-D; synthesis of DOX-D; CD spectra of pH-responsive hydrogel; characterization of hydrogel membranes. See DOI: 10.1039/c6sc04770jClick here for additional data file.

    doi: 10.1039/c6sc04770j

    Figure Lengend Snippet: (A) Time-dependent release of TMR-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). (B) Fluorescence spectra corresponding to the release of TMR-D upon exposing the microcapsules loaded with TMR-D to various concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50 and (f) 75 mM. Inset: derived calibration curve corresponding to the fluorescence intensities of the released TMR-D at λ em = 582 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of TMR-D upon the treatment of the ATP-responsive microcapsules with different nucleotide triphosphates for a fixed time interval of 30 min: (a) 25 mM ATP, (b) 25 mM TTP, (c) 25 mM CTP, (d) 25 mM GTP, and (e) untreated.

    Article Snippet: Reagents and materials Magnesium chloride, sodium chloride, doxorubicin hydrochloride (DOX), 4-carboxyphenylboronic acid, dextran (MW = 40 kDa), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES base), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES acid), N -(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDC), N -hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (MW = 30, 450, and 1250 kDa), 2-(N -morpholino)ethanesulfonic acid (MES), ammonium persulfate (APS), N ,N ,N ′,N ′-tetramethylethylenediamine (TEMED), acrylamide solution (40%), poly(allylamine hydrochloride) (PAH, MW = 58 kDa), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP) were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Derivative Assay

    Distribution of FtsZ in the PEG/dextran 500 LLPS system. ( a ) Representative confocal images of FtsZ within the PEG/dextran 500 (3:1) emulsions in the absence and presence of 1 mM GTP. Total FtsZ concentration was 7 μM. A schematic illustration of the disposition of FtsZ within the phases is depicted on the right. ( b ) Concentration dependence of the distribution of FtsZ within the mixture as determined by fluorescence, together with an illustration on the right, in the absence and presence of 1 mM GTP. Data are the average of at least 3 independent measurements ± SD.

    Journal: Scientific Reports

    Article Title: Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    doi: 10.1038/srep35140

    Figure Lengend Snippet: Distribution of FtsZ in the PEG/dextran 500 LLPS system. ( a ) Representative confocal images of FtsZ within the PEG/dextran 500 (3:1) emulsions in the absence and presence of 1 mM GTP. Total FtsZ concentration was 7 μM. A schematic illustration of the disposition of FtsZ within the phases is depicted on the right. ( b ) Concentration dependence of the distribution of FtsZ within the mixture as determined by fluorescence, together with an illustration on the right, in the absence and presence of 1 mM GTP. Data are the average of at least 3 independent measurements ± SD.

    Article Snippet: Reagents Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma.

    Techniques: Concentration Assay, Fluorescence

    Distribution of FtsZ in the PEG/DNA LLPS system. ( a ) Representative confocal images of FtsZ within the PEG/DNA (3:1) emulsions in the absence and presence of 1 mM GTP. Total FtsZ concentration was 12 (GDP) and 8 μM (GTP). A schematic illustration of the disposition of FtsZ within the phases is depicted on the right. ( b ) Concentration dependence of the distribution of FtsZ-GDP within the mixture as determined by fluorescence together with an illustration on the right. Data are the average of at least 3 independent measurements ± SD.

    Journal: Scientific Reports

    Article Title: Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    doi: 10.1038/srep35140

    Figure Lengend Snippet: Distribution of FtsZ in the PEG/DNA LLPS system. ( a ) Representative confocal images of FtsZ within the PEG/DNA (3:1) emulsions in the absence and presence of 1 mM GTP. Total FtsZ concentration was 12 (GDP) and 8 μM (GTP). A schematic illustration of the disposition of FtsZ within the phases is depicted on the right. ( b ) Concentration dependence of the distribution of FtsZ-GDP within the mixture as determined by fluorescence together with an illustration on the right. Data are the average of at least 3 independent measurements ± SD.

    Article Snippet: Reagents Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma.

    Techniques: Concentration Assay, Fluorescence

    Distribution of FtsZ in the PEG/Ficoll 70 LLPS system. ( a ) Representative confocal images of FtsZ within the PEG/Ficoll (3:1) emulsions in the absence and presence of 1 mM GTP. Total FtsZ concentration was 7 μM. A schematic illustration of the disposition of FtsZ within the phases is depicted on the right. ( b ) Concentration dependence of the distribution of FtsZ within the mixture as determined by fluorescence, together with an illustration on the right, in the absence and presence of 1 mM GTP. Data are the average of at least 3 independent measurements ± SD.

    Journal: Scientific Reports

    Article Title: Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    doi: 10.1038/srep35140

    Figure Lengend Snippet: Distribution of FtsZ in the PEG/Ficoll 70 LLPS system. ( a ) Representative confocal images of FtsZ within the PEG/Ficoll (3:1) emulsions in the absence and presence of 1 mM GTP. Total FtsZ concentration was 7 μM. A schematic illustration of the disposition of FtsZ within the phases is depicted on the right. ( b ) Concentration dependence of the distribution of FtsZ within the mixture as determined by fluorescence, together with an illustration on the right, in the absence and presence of 1 mM GTP. Data are the average of at least 3 independent measurements ± SD.

    Article Snippet: Reagents Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma.

    Techniques: Concentration Assay, Fluorescence

    Distribution of FtsZ polymers in several binary mixtures encircled by a lipid layer. ( a ) PEG/dextran 500 (3:1), total FtsZ concentration 7 μM. ( b ) PEG/Ficoll (1:1), total FtsZ concentration 6 μM. ( c ) PEG/DNA (1:1), total FtsZ concentration 12 μM. In all the images of the superimposed channels, lines depict the region through which the intensity profiles for each individual channel (red curve for Alexa 647, green curve for FITC or Alexa 488) were obtained. 1 mM GTP. Cartoons represent the distribution of FtsZ within the encircled phases.

    Journal: Scientific Reports

    Article Title: Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    doi: 10.1038/srep35140

    Figure Lengend Snippet: Distribution of FtsZ polymers in several binary mixtures encircled by a lipid layer. ( a ) PEG/dextran 500 (3:1), total FtsZ concentration 7 μM. ( b ) PEG/Ficoll (1:1), total FtsZ concentration 6 μM. ( c ) PEG/DNA (1:1), total FtsZ concentration 12 μM. In all the images of the superimposed channels, lines depict the region through which the intensity profiles for each individual channel (red curve for Alexa 647, green curve for FITC or Alexa 488) were obtained. 1 mM GTP. Cartoons represent the distribution of FtsZ within the encircled phases.

    Article Snippet: Reagents Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma.

    Techniques: Concentration Assay

    Overview of the biological system and experimental procedure. ( a ) Scheme of the association reactions of FtsZ. When bound to GDP, FtsZ is found as an ensemble of species of small size 16 . Upon GTP binding, and above the critical concentration of polymerization, FtsZ self assembles forming one subunit thick polymers of a defined size 59 60 , that in the presence of crowding agents interact laterally forming higher order structures 22 23 24 . Depletion of the nucleotide by FtsZ GTPase activity increases the ratio of GDP, and the protein depolymerizes. ( b ) Experimental approaches followed. Left, LLPS systems with labelled FtsZ were centrifuged after thoroughly mixing. Both phases separate, the PEG-rich one being always located in the top part of the solution. The fluorescence in each phase is measured allowing quantification of FtsZ concentration. Qualitative distribution and organization of FtsZ was assessed by microscopy using bulk LLPS emulsions (middle) and LLPS emulsions encapsulated in lipid coated microdroplets (right). Each of these approaches was done with the three different LLPS compositions described in the main text.

    Journal: Scientific Reports

    Article Title: Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    doi: 10.1038/srep35140

    Figure Lengend Snippet: Overview of the biological system and experimental procedure. ( a ) Scheme of the association reactions of FtsZ. When bound to GDP, FtsZ is found as an ensemble of species of small size 16 . Upon GTP binding, and above the critical concentration of polymerization, FtsZ self assembles forming one subunit thick polymers of a defined size 59 60 , that in the presence of crowding agents interact laterally forming higher order structures 22 23 24 . Depletion of the nucleotide by FtsZ GTPase activity increases the ratio of GDP, and the protein depolymerizes. ( b ) Experimental approaches followed. Left, LLPS systems with labelled FtsZ were centrifuged after thoroughly mixing. Both phases separate, the PEG-rich one being always located in the top part of the solution. The fluorescence in each phase is measured allowing quantification of FtsZ concentration. Qualitative distribution and organization of FtsZ was assessed by microscopy using bulk LLPS emulsions (middle) and LLPS emulsions encapsulated in lipid coated microdroplets (right). Each of these approaches was done with the three different LLPS compositions described in the main text.

    Article Snippet: Reagents Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma.

    Techniques: Binding Assay, Concentration Assay, Activity Assay, Fluorescence, Microscopy

    Dynamic behaviour of FtsZ polymers within LLPS systems inside lipid containers. ( a ) PEG/dextran 500 (3:1), total FtsZ concentration 6 μM. Monitoring of depolymerization started 14 min after GTP addition. ( b ) PEG/DNA (1:1), total FtsZ concentration 12 μM. Monitoring of depolymerization started ∼2 h 20 min after GTP addition. In all panels, distribution of FtsZ at the specified time in minutes, time zero being the beginning of visualisation. 0.75 mM GTP. Fluorescence signals correspond to PEG-Alexa 647 (red) and FtsZ-Alexa 488 (green). Lines indicate the region, adapted in each image depending on the orientation of the droplet, through which the intensity profiles below depicting the distribution of FtsZ were obtained (black and grey, zero and end times, respectively). Random variations of the profiles due to subtle movements on the droplet during visualisation were corrected by matching maxima corresponding to the lipid boundary along the x axis.

    Journal: Scientific Reports

    Article Title: Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    doi: 10.1038/srep35140

    Figure Lengend Snippet: Dynamic behaviour of FtsZ polymers within LLPS systems inside lipid containers. ( a ) PEG/dextran 500 (3:1), total FtsZ concentration 6 μM. Monitoring of depolymerization started 14 min after GTP addition. ( b ) PEG/DNA (1:1), total FtsZ concentration 12 μM. Monitoring of depolymerization started ∼2 h 20 min after GTP addition. In all panels, distribution of FtsZ at the specified time in minutes, time zero being the beginning of visualisation. 0.75 mM GTP. Fluorescence signals correspond to PEG-Alexa 647 (red) and FtsZ-Alexa 488 (green). Lines indicate the region, adapted in each image depending on the orientation of the droplet, through which the intensity profiles below depicting the distribution of FtsZ were obtained (black and grey, zero and end times, respectively). Random variations of the profiles due to subtle movements on the droplet during visualisation were corrected by matching maxima corresponding to the lipid boundary along the x axis.

    Article Snippet: Reagents Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma.

    Techniques: Concentration Assay, Fluorescence

    Binding of KIF1A to the microtubule lattice is negatively affected by GTP-tubulin and EB proteins, but not tubulin tyrosination. A) Binding and B) quantification of KIF1A, KIF5B, and KIF5C to GMPCPP- and GDP-taxol-stabilized microtubules in the presence of AMP-PNP (n=1004-5855 microtubules per condition; n.s., non-significant, ***p

    Journal: Current biology : CB

    Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

    doi: 10.1016/j.cub.2018.11.065

    Figure Lengend Snippet: Binding of KIF1A to the microtubule lattice is negatively affected by GTP-tubulin and EB proteins, but not tubulin tyrosination. A) Binding and B) quantification of KIF1A, KIF5B, and KIF5C to GMPCPP- and GDP-taxol-stabilized microtubules in the presence of AMP-PNP (n=1004-5855 microtubules per condition; n.s., non-significant, ***p

    Article Snippet: GDP-taxol-stabilized microtubules were prepared by incubating unlabeled soluble tubulin with 5% HyLite-647-labeled tubulin (Cytoskeleton) in BRB80 + 1 mM Mg-GTP (Sigma) and 30 μM taxol (paclitaxel; Cytoskeleton) for 30 minutes at 37°C.

    Techniques: Binding Assay

    UL42 is transported into the nucleus via the importin α/β pathway. (A) UL42 competes with the SV40 TAg–NLS, but not with the hnRNP A1-M9, for nuclear import. Nuclear import was performed in the presence of RRL, an ATP-regenerating system, and purified Flag–UL42 (+). TAg–NLS, 1 mM SV40 TAg–NLS peptide was added to the import mixture. A1-M9, 1 mM hnRNP A1-M9 peptide was added to the import mixture. The merged FITC and DAPI signals are shown. Images are representative of three independent nuclear import assays. (B) GTP dependence of UL42 nuclear uptake. Nuclear import was performed in the presence of RRL, an ATP-regenerating system, and purified Flag–UL42 (+). GTPγS, GTP was omitted and 1 mM GTPγS was added. The merged FITC and DAPI signals are shown. Images are representative of three independent nuclear import assays. (C) UL42 gains entry to the nucleus in the presence of importins α and β. Reconstitution assays were performed in transport buffer (TB), with a Ran mixture (3 μM Ran and 0.5 μM NTF2) (Ran), and the addition of 1 μM purified importin α4 (α) or 1 μM importin β (β) or 1 μM concentrations of both importin α4 and importin β (α+β), with 200 μg/ml purified Flag–UL42 as the import substrate. The merged FITC and DAPI signals are shown. Images are representative of three independent nuclear import assays.

    Journal: Frontiers in Microbiology

    Article Title: The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

    doi: 10.3389/fmicb.2016.00124

    Figure Lengend Snippet: UL42 is transported into the nucleus via the importin α/β pathway. (A) UL42 competes with the SV40 TAg–NLS, but not with the hnRNP A1-M9, for nuclear import. Nuclear import was performed in the presence of RRL, an ATP-regenerating system, and purified Flag–UL42 (+). TAg–NLS, 1 mM SV40 TAg–NLS peptide was added to the import mixture. A1-M9, 1 mM hnRNP A1-M9 peptide was added to the import mixture. The merged FITC and DAPI signals are shown. Images are representative of three independent nuclear import assays. (B) GTP dependence of UL42 nuclear uptake. Nuclear import was performed in the presence of RRL, an ATP-regenerating system, and purified Flag–UL42 (+). GTPγS, GTP was omitted and 1 mM GTPγS was added. The merged FITC and DAPI signals are shown. Images are representative of three independent nuclear import assays. (C) UL42 gains entry to the nucleus in the presence of importins α and β. Reconstitution assays were performed in transport buffer (TB), with a Ran mixture (3 μM Ran and 0.5 μM NTF2) (Ran), and the addition of 1 μM purified importin α4 (α) or 1 μM importin β (β) or 1 μM concentrations of both importin α4 and importin β (α+β), with 200 μg/ml purified Flag–UL42 as the import substrate. The merged FITC and DAPI signals are shown. Images are representative of three independent nuclear import assays.

    Article Snippet: The ATP dependence of importation was tested by replacing ATP with 1 mM 5′-adenylimidodiphosphate (AMP–PNP; Sigma), and the GTP dependence was tested by adding 1 mM guanosine 5′-O -(3-thiotriphosphate) (GTPγS; Sigma) in place of GTP.

    Techniques: Purification

    DRP1 ring- and spiral-like structures by negative-stain TEM. Negative-stained TEM images of DRP1 in the absence of nucleotide (A) or in the presence of GDP (B), GMP-PNP (C), or GTP (D). Scale bar = 50 nm).

    Journal: PLoS ONE

    Article Title: Molecular mechanism of DRP1 assembly studied in vitro by cryo-electron microscopy

    doi: 10.1371/journal.pone.0179397

    Figure Lengend Snippet: DRP1 ring- and spiral-like structures by negative-stain TEM. Negative-stained TEM images of DRP1 in the absence of nucleotide (A) or in the presence of GDP (B), GMP-PNP (C), or GTP (D). Scale bar = 50 nm).

    Article Snippet: Incubation was typically performed for 4 hours on ice at a DRP1 concentration of 2.3 mg/mL with 2 mM MgCl2 in the absence or presence of 2 mM nucleotide GTP, GDP, or GMP-PNP (Sigma-Aldrich).

    Techniques: Staining, Transmission Electron Microscopy

    Resealing of semi-intact HeLa cells by the addition of CaCl 2 . A. Basic protocol for the resealing of semi-intact HeLa cells with CaCl 2 . B. HeLa cells were permeabilized with SLO, and were incubated with 1.5 mg/ml L5178Y cytosol, an ATP regenerating system, GTP, glucose, and fluorescein-conjugated dextran (ATP/GTP/glucose/fluorescein-dextran) at 32°C for 15 min. The cells were incubated for a further 5 min at 32°C with or without 1 mM CaCl 2 . Resealed (CaCl 2 -treated) or semi-intact (CaCl 2 -untreated) cells were incubated with DMEM supplemented with 10% FCS or TB at 37°C for 30 min. The cells were observed under a confocal microscope. Staining with both fluorescein-dextran (green) and PI (red) indicated that the cells had been resealed efficiently. Semi-intact cells were positive for PI staining but not for fluorescein-dextran. Bar = 20 µm. C. Semi-intact HeLa cells were incubated with 0.0, 1.5, 3.0, 5.0, or 7.0 mg/ml L5178Y cytosol and ATP/GTP/glucose/fluorescein-dextran at 32°C for 15 min, and then were resealed as described in A. Means and standard deviations for the percentage of resealed cells, which were stained with both PI and fluorescein-dextran, are shown in the graph. D. Semi-intact HeLa cells were incubated with L5178Y cytosol as described in A, and then were resealed by treatment with 0.0, 0.1, 0.5, 1.0, or 3.0 mM CaCl 2 at 32°C for 5 min. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph. E. Semi-intact HeLa cells were incubated with L5178Y cytosol as described in A, and then were resealed by treatment with 1 mM CaCl 2 at 32°C for 0.0, 1.5, 3.0, 5.0, or 7.5 min. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph. F. Semi-intact HeLa cells were incubated with 1.5 mg/ml L5178Y cytosol and ATP/GTP/glucose/fluorescein-dextran at 32°C for 15, 30, 60, or 90 min, and then were resealed as described in A. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph.

    Journal: PLoS ONE

    Article Title: A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

    doi: 10.1371/journal.pone.0044127

    Figure Lengend Snippet: Resealing of semi-intact HeLa cells by the addition of CaCl 2 . A. Basic protocol for the resealing of semi-intact HeLa cells with CaCl 2 . B. HeLa cells were permeabilized with SLO, and were incubated with 1.5 mg/ml L5178Y cytosol, an ATP regenerating system, GTP, glucose, and fluorescein-conjugated dextran (ATP/GTP/glucose/fluorescein-dextran) at 32°C for 15 min. The cells were incubated for a further 5 min at 32°C with or without 1 mM CaCl 2 . Resealed (CaCl 2 -treated) or semi-intact (CaCl 2 -untreated) cells were incubated with DMEM supplemented with 10% FCS or TB at 37°C for 30 min. The cells were observed under a confocal microscope. Staining with both fluorescein-dextran (green) and PI (red) indicated that the cells had been resealed efficiently. Semi-intact cells were positive for PI staining but not for fluorescein-dextran. Bar = 20 µm. C. Semi-intact HeLa cells were incubated with 0.0, 1.5, 3.0, 5.0, or 7.0 mg/ml L5178Y cytosol and ATP/GTP/glucose/fluorescein-dextran at 32°C for 15 min, and then were resealed as described in A. Means and standard deviations for the percentage of resealed cells, which were stained with both PI and fluorescein-dextran, are shown in the graph. D. Semi-intact HeLa cells were incubated with L5178Y cytosol as described in A, and then were resealed by treatment with 0.0, 0.1, 0.5, 1.0, or 3.0 mM CaCl 2 at 32°C for 5 min. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph. E. Semi-intact HeLa cells were incubated with L5178Y cytosol as described in A, and then were resealed by treatment with 1 mM CaCl 2 at 32°C for 0.0, 1.5, 3.0, 5.0, or 7.5 min. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph. F. Semi-intact HeLa cells were incubated with 1.5 mg/ml L5178Y cytosol and ATP/GTP/glucose/fluorescein-dextran at 32°C for 15, 30, 60, or 90 min, and then were resealed as described in A. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph.

    Article Snippet: Reagents and Antibodies GTP, ATP, creatine phosphate, and creatine kinase were obtained from Sigma.

    Techniques: Incubation, Microscopy, Staining

    Introduction of fluorescein-dextran of different molecular weights into resealed cells. A. HeLa cells were incubated with or without (2000 kDa dextran w/o SLO) 0.13 µg/ml SLO on ice for 5 min. After wash with PBS three times, the cells were further with transport buffer containing propidium iodide at 32°C for 5 min. Semi-intact HeLa cells were incubated with 1.5 mg/ml L5178Y cytosol, an ATP regenerating system, GTP, glucose, and 100 µg/ml fluorescein-dextran of 3, 10, 40, 70, or 2000 kDa at 32°C for 15 min, and then were resealed by treatment with 1 mM CaCl 2 at 32°C for 5 min. After incubation with DMEM supplemented with FCS for 30 min, the cells were observed by confocal microscopy. Since the cells without SLO treatment did not contain the fluorescence of propidium iodide, differential interference contrast (DIC) image was shown. Bar = 10 µm. B. HeLa cells were treated as described in A, were trypsinized, and were subjected to flowcytometry. The histograms of fluorescein fluorescence of dextran with different molecular weight in PI-positive cells were shown.

    Journal: PLoS ONE

    Article Title: A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

    doi: 10.1371/journal.pone.0044127

    Figure Lengend Snippet: Introduction of fluorescein-dextran of different molecular weights into resealed cells. A. HeLa cells were incubated with or without (2000 kDa dextran w/o SLO) 0.13 µg/ml SLO on ice for 5 min. After wash with PBS three times, the cells were further with transport buffer containing propidium iodide at 32°C for 5 min. Semi-intact HeLa cells were incubated with 1.5 mg/ml L5178Y cytosol, an ATP regenerating system, GTP, glucose, and 100 µg/ml fluorescein-dextran of 3, 10, 40, 70, or 2000 kDa at 32°C for 15 min, and then were resealed by treatment with 1 mM CaCl 2 at 32°C for 5 min. After incubation with DMEM supplemented with FCS for 30 min, the cells were observed by confocal microscopy. Since the cells without SLO treatment did not contain the fluorescence of propidium iodide, differential interference contrast (DIC) image was shown. Bar = 10 µm. B. HeLa cells were treated as described in A, were trypsinized, and were subjected to flowcytometry. The histograms of fluorescein fluorescence of dextran with different molecular weight in PI-positive cells were shown.

    Article Snippet: Reagents and Antibodies GTP, ATP, creatine phosphate, and creatine kinase were obtained from Sigma.

    Techniques: Incubation, Confocal Microscopy, Fluorescence, Molecular Weight

    Intracellular PI3P was decreased in a p38 MAPK-dependent manner in Db cells. A. HeLa cells were pretreated with or without 2 µM SB203580 or 2 µM SB202190 at 37°C for 60 min. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol, an ATP regenerating system, GTP, and glucose in the presence or absence of 2 µM SB203580 or SB202190 at 32°C for 30 min, and then for a further 15 min at 32°C after the addition of 1 µg of GST-2xFYVE recombinant protein. The cells were fixed and GST-2xFYVE was visualized with Alexa488-conjugated antibodies against GST. Bar = 10 µm. B. The fluorescence intensity of GST-2xFYVE was measured as described in Materials and Methods, and the means and standard deviations for the fluorescence intensity are shown in the graph. We performed three independent experiments and counted 100 cells in each experiment. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the P value was

    Journal: PLoS ONE

    Article Title: A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

    doi: 10.1371/journal.pone.0044127

    Figure Lengend Snippet: Intracellular PI3P was decreased in a p38 MAPK-dependent manner in Db cells. A. HeLa cells were pretreated with or without 2 µM SB203580 or 2 µM SB202190 at 37°C for 60 min. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol, an ATP regenerating system, GTP, and glucose in the presence or absence of 2 µM SB203580 or SB202190 at 32°C for 30 min, and then for a further 15 min at 32°C after the addition of 1 µg of GST-2xFYVE recombinant protein. The cells were fixed and GST-2xFYVE was visualized with Alexa488-conjugated antibodies against GST. Bar = 10 µm. B. The fluorescence intensity of GST-2xFYVE was measured as described in Materials and Methods, and the means and standard deviations for the fluorescence intensity are shown in the graph. We performed three independent experiments and counted 100 cells in each experiment. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the P value was

    Article Snippet: Reagents and Antibodies GTP, ATP, creatine phosphate, and creatine kinase were obtained from Sigma.

    Techniques: Incubation, Recombinant, Fluorescence

    Retrograde transport of Cholera toxin B subunit (CtxB) in WT and Db cells. A. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol in the presence of ATP/GTP/glucose and Alexa647-conjugated dextran (10 kDa, blue) at 32°C for 30 min, and then resealed by addition of 1 mM CaCl 2 for 5 min. After incubation with DMEM supplemented with 10% FCS for 30 min, the cells were treated with 2 µg/ml Alexa546-conjugated CtxB (red) on ice for 30 min, and then incubated with medium at 37°C for 0, 15, 30, and 45 min. The cells were fixed, were immunostained with antibodies against GM130 (green), and were observed by confocal microscope. Bar = 10 µm. B. We counted the number of cells in which CtxB was accumulated at the Golgi on the basis of the colocalization of CtxB with GM130, after a 0, 15, 30, and 45 min chase in WT (○) and Db (•) cells with or without treatment of 2 µM SB203580 (△, WT+SB; ▴, Db+SB). The means and standard deviations for the percentages of these cells are shown in the graph. Three independent experiments were performed and we counted 300 cells in each experiment. C. CtxB transport assay was performed in WT or Db cells treated with or without 2 µM SB203580 or 2 µM SB202190. In 30 or 45 min after internalization of CtxB by incubating cells at 37°C, the cells were fixed and the number of cells in which CtxB was accumulated at the Golgi was counted. The means and standard deviations for the percentages of the cells are shown in the graph. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the P value was

    Journal: PLoS ONE

    Article Title: A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

    doi: 10.1371/journal.pone.0044127

    Figure Lengend Snippet: Retrograde transport of Cholera toxin B subunit (CtxB) in WT and Db cells. A. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol in the presence of ATP/GTP/glucose and Alexa647-conjugated dextran (10 kDa, blue) at 32°C for 30 min, and then resealed by addition of 1 mM CaCl 2 for 5 min. After incubation with DMEM supplemented with 10% FCS for 30 min, the cells were treated with 2 µg/ml Alexa546-conjugated CtxB (red) on ice for 30 min, and then incubated with medium at 37°C for 0, 15, 30, and 45 min. The cells were fixed, were immunostained with antibodies against GM130 (green), and were observed by confocal microscope. Bar = 10 µm. B. We counted the number of cells in which CtxB was accumulated at the Golgi on the basis of the colocalization of CtxB with GM130, after a 0, 15, 30, and 45 min chase in WT (○) and Db (•) cells with or without treatment of 2 µM SB203580 (△, WT+SB; ▴, Db+SB). The means and standard deviations for the percentages of these cells are shown in the graph. Three independent experiments were performed and we counted 300 cells in each experiment. C. CtxB transport assay was performed in WT or Db cells treated with or without 2 µM SB203580 or 2 µM SB202190. In 30 or 45 min after internalization of CtxB by incubating cells at 37°C, the cells were fixed and the number of cells in which CtxB was accumulated at the Golgi was counted. The means and standard deviations for the percentages of the cells are shown in the graph. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the P value was

    Article Snippet: Reagents and Antibodies GTP, ATP, creatine phosphate, and creatine kinase were obtained from Sigma.

    Techniques: Incubation, Microscopy, Transport Assay

    Mutational analyses of basic residues in cpFtsY. A, representative stimulated GTPase reactions of cpSRP54 with wild-type cpFtsY ( black ) and mutants cpFtsY(K191D/K193D) ( blue ), cpFtsY(K203D/R204D) ( purple ), and cpFtsY(K234D/K235D) ( green . B, basal GTP hydrolysis rate constants for wild-type and mutant cpFtsY, determined as described under “Experimental Procedures.” C, fluorescence emission spectra for acrylodan-labeled cpSRP54 by itself (○) and in the presence of wild-type cpFtsY (●) or mutant cpFtsY(K203D/R204D) ( purple ).

    Journal: The Journal of Biological Chemistry

    Article Title: Co-evolution of Two GTPases Enables Efficient Protein Targeting in an RNA-less Chloroplast Signal Recognition Particle Pathway *

    doi: 10.1074/jbc.M116.752931

    Figure Lengend Snippet: Mutational analyses of basic residues in cpFtsY. A, representative stimulated GTPase reactions of cpSRP54 with wild-type cpFtsY ( black ) and mutants cpFtsY(K191D/K193D) ( blue ), cpFtsY(K203D/R204D) ( purple ), and cpFtsY(K234D/K235D) ( green . B, basal GTP hydrolysis rate constants for wild-type and mutant cpFtsY, determined as described under “Experimental Procedures.” C, fluorescence emission spectra for acrylodan-labeled cpSRP54 by itself (○) and in the presence of wild-type cpFtsY (●) or mutant cpFtsY(K203D/R204D) ( purple ).

    Article Snippet: To form the GTP-bound cpSRP54·cpFtsY or cpSRP54-NG·cpFtsY complex, 2 m m GTP (Sigma) was used; GDP released during the course of the reaction was minimal, as explained previously ( ).

    Techniques: Mutagenesis, Fluorescence, Labeling

    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active (Rac1-GTP) and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p

    Journal: PLoS ONE

    Article Title: Aberrant Glycogen Synthase Kinase 3? Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

    doi: 10.1371/journal.pone.0055289

    Figure Lengend Snippet: Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active (Rac1-GTP) and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p

    Article Snippet: The fraction of Rac1 bound to guanosine triphosphate (GTP) (Rac1-GTP, an active form) was eluted from the resins and detected by Western blot analysis using rabbit polyclonal antibody to Rac1 (diluted 1∶1,000; Thermo).

    Techniques: Inhibition, Expressing, Wound Healing Assay, Pull Down Assay, Western Blot, Zymography, Quantitative RT-PCR

    The release of inorganic phosphate (Pi) by GTP hydrolysis through an interaction between TBC1D15 and Rab7a-WT. The TBC1D15 protein shows Rab-GAP activity for Rab. GTP was used as the substrate for Rab. The blank control (buffer) was the only sample without any protein. The release of Pi was detected using a spectrophotometer at 360 nm (A 360 ) every 5 min.

    Journal: Marine Drugs

    Article Title: A New Member of the TBC1D15 Family from Chiloscyllium plagiosum: Rab GTPase-Activating Protein Based on Rab7 as a Substrate

    doi: 10.3390/md13052955

    Figure Lengend Snippet: The release of inorganic phosphate (Pi) by GTP hydrolysis through an interaction between TBC1D15 and Rab7a-WT. The TBC1D15 protein shows Rab-GAP activity for Rab. GTP was used as the substrate for Rab. The blank control (buffer) was the only sample without any protein. The release of Pi was detected using a spectrophotometer at 360 nm (A 360 ) every 5 min.

    Article Snippet: To further verify the changes of GTP hydrolysis due to GAP activity, we used a phosphate assay kit (EnzChek® Phosphate Assay Kit, Invitrogen, E6646, Eugene, OR, USA) to detect the Pi content with GTP-GDP exchange.

    Techniques: Activity Assay, Spectrophotometry

    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both ATP and GTP (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both ATP and GTP (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Incubation, Luciferase, Activity Assay

    In vitro homotypic ER fusion requires the atlastin GTPase Sey1p. (A) GTP hydrolysis is required for ER fusion in vitro. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of the indicated nucleotide for 90 min. Fusion values were normalized to those obtained using an ATP/GTP-driven reaction incubated at 27°C. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: In vitro homotypic ER fusion requires the atlastin GTPase Sey1p. (A) GTP hydrolysis is required for ER fusion in vitro. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of the indicated nucleotide for 90 min. Fusion values were normalized to those obtained using an ATP/GTP-driven reaction incubated at 27°C. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: In Vitro, Incubation

    Sey1p at its physiological concentration is not sufficient to induce liposome fusion. (A) Proteoliposomes with the indicated Sey1p-to-lipid ratio were generated as described in the Materials and methods. Donor and acceptor proteoliposomes were mixed and incubated at 30°C for 10 min. After GTP and Mg 2+ were added, and NBD fluorescence was measured at 30-s intervals for 30 min. β-Octylglucoside was then added to determine total fluorescence. Fusion is expressed as the percentage of total fluorescence. All experiments were performed multiple times with similar results, and the data shown are representative of all results. (B) Overexpression of Sey1p restores fusion to sec22 Δ microsomes. The indicated microsomes were incubated on ice or at 27°C with GTP/ATP in the absence or presence of anti-Sey1p antibodies for 90 min. SEY1-OE , SEY1 overexpressor. Data represent the means ± SEM (error bars; n = 3). ***, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Sey1p at its physiological concentration is not sufficient to induce liposome fusion. (A) Proteoliposomes with the indicated Sey1p-to-lipid ratio were generated as described in the Materials and methods. Donor and acceptor proteoliposomes were mixed and incubated at 30°C for 10 min. After GTP and Mg 2+ were added, and NBD fluorescence was measured at 30-s intervals for 30 min. β-Octylglucoside was then added to determine total fluorescence. Fusion is expressed as the percentage of total fluorescence. All experiments were performed multiple times with similar results, and the data shown are representative of all results. (B) Overexpression of Sey1p restores fusion to sec22 Δ microsomes. The indicated microsomes were incubated on ice or at 27°C with GTP/ATP in the absence or presence of anti-Sey1p antibodies for 90 min. SEY1-OE , SEY1 overexpressor. Data represent the means ± SEM (error bars; n = 3). ***, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Concentration Assay, Generated, Incubation, Fluorescence, Over Expression

    ER SNAREs are involved in Sey1p-dependent ER fusion. (A) Sec22p is involved in Sey1p-mediated ER fusion. The indicated microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups. A Tukey’s test between reactions was performed at 27°C. P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: ER SNAREs are involved in Sey1p-dependent ER fusion. (A) Sec22p is involved in Sey1p-mediated ER fusion. The indicated microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups. A Tukey’s test between reactions was performed at 27°C. P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Incubation

    In vitro homotypic ER fusion is Rab independent. (A) Homotypic ER fusion is insensitive to Rab inhibition by Gdi1p/Gyp1p. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C with ATP/GTP in the absence or presence of his 6 -Gdi1p and his 6 -Gyp1p for 90 min. For 1×Gdi1p/Gyp1p, the concentrations of his 6 -Gdi1p and his 6 -Gyp1p were 1.5 and 2.5 µM, respectively; the corresponding concentrations for 4×Gdi1p/Gyp1p were 6 and 10 µM, respectively. Data represent the means ± SEM (error bars; n = 3). ***, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: In vitro homotypic ER fusion is Rab independent. (A) Homotypic ER fusion is insensitive to Rab inhibition by Gdi1p/Gyp1p. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C with ATP/GTP in the absence or presence of his 6 -Gdi1p and his 6 -Gyp1p for 90 min. For 1×Gdi1p/Gyp1p, the concentrations of his 6 -Gdi1p and his 6 -Gyp1p were 1.5 and 2.5 µM, respectively; the corresponding concentrations for 4×Gdi1p/Gyp1p were 6 and 10 µM, respectively. Data represent the means ± SEM (error bars; n = 3). ***, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: In Vitro, Inhibition, Incubation

    The SNARE chaperones Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion. (A) Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion, but not in GTP-driven fusion. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Some reactions were treated with anti-Sec17p antibodies, anti-Sec18p antibodies, or recombinant his 6 -Sec18p at the indicated concentrations. Data represent the means ± SEM (error bars; n = 3). **, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: The SNARE chaperones Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion. (A) Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion, but not in GTP-driven fusion. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Some reactions were treated with anti-Sec17p antibodies, anti-Sec18p antibodies, or recombinant his 6 -Sec18p at the indicated concentrations. Data represent the means ± SEM (error bars; n = 3). **, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Incubation, Recombinant

    Development of an in vitro homotypic ER fusion assay. (A) Schematic representation of chimeric proteins for the Gluc PCA. (B) Assay scheme; see Results for details. Microsomes isolated from BJ-Gluc1 yeast cells overexpressing ssZIP-Gluc1-HDEL under the control of an ADH1 promoter (Gluc1 microsomes) were mixed with microsomes isolated from BJ-Gluc2 yeast cells overexpressing ssZIP-Gluc2-HDEL (Gluc2 microsomes) and then incubated at 27°C in the presence of GTP and ATP. After 90 min, the luciferase substrate coelenterazine was added, and luciferase activity was measured. Excess GST-ZIP was added to block extra-luminal reconstitution of functional Gluc caused by membrane destabilization or rupture during incubation. (C) Expression of Gluc PCA fragments in isolated microsomes. The expression of Gluc PCA fragments was analyzed by immunoblotting using the indicated antibodies. Kar2p, an ER-resident protein, was used as a loading control. (D) GTP-driven homotypic ER fusion is markedly enhanced by ATP. Gluc1 and Gluc2 microsomes were mixed and incubated on ice or at 27°C in the presence of ATP and/or GTP for 90 min. Data represent means ± SEM (error bars; n = 3). RLU, relative luminescence unit. ***, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Development of an in vitro homotypic ER fusion assay. (A) Schematic representation of chimeric proteins for the Gluc PCA. (B) Assay scheme; see Results for details. Microsomes isolated from BJ-Gluc1 yeast cells overexpressing ssZIP-Gluc1-HDEL under the control of an ADH1 promoter (Gluc1 microsomes) were mixed with microsomes isolated from BJ-Gluc2 yeast cells overexpressing ssZIP-Gluc2-HDEL (Gluc2 microsomes) and then incubated at 27°C in the presence of GTP and ATP. After 90 min, the luciferase substrate coelenterazine was added, and luciferase activity was measured. Excess GST-ZIP was added to block extra-luminal reconstitution of functional Gluc caused by membrane destabilization or rupture during incubation. (C) Expression of Gluc PCA fragments in isolated microsomes. The expression of Gluc PCA fragments was analyzed by immunoblotting using the indicated antibodies. Kar2p, an ER-resident protein, was used as a loading control. (D) GTP-driven homotypic ER fusion is markedly enhanced by ATP. Gluc1 and Gluc2 microsomes were mixed and incubated on ice or at 27°C in the presence of ATP and/or GTP for 90 min. Data represent means ± SEM (error bars; n = 3). RLU, relative luminescence unit. ***, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: In Vitro, Single Vesicle Fusion Assay, Isolation, Incubation, Luciferase, Activity Assay, Blocking Assay, Functional Assay, Expressing

    Sey1p physically interacts with ER SNAREs. (A and B) Sey1p physically interacts with the ER SNARE proteins Sec22p and Ufe1p. Microsomes isolated from BJ3505 were detergent-solubilized and incubated with anti-Sec22p antibodies (A), anti-Ufe1p antibodies (B), or preimmune IgG (control) in the presence of protein A Sepharose. Protein A Sepharose–bound material was then analyzed by immunoblotting using the indicated antibodies. (C) Sec18p regulates the interaction between Sec22p and Sey1p. BJ3505 microsomes were preincubated in the absence or presence of anti-Sec18p antibodies for 10 min at 4°C. After ATP/GTP was added, microsomes were further incubated for 40 min. During incubation, some samples received anti-Sec18p antibodies at the indicated time points. Sec22p was precipitated using anti-Sec22p antibody–conjugated Dynabeads, and bound proteins were analyzed by immunoblotting using the indicated antibodies. (D) Detection of an in vivo interaction between Sec22p and Sey1p by DSP cross-linking. BJ3505 spheroplasts were incubated in the absence or presence of 4 mM DSP for 30 min at 4°C. After DSP was quenched, detergent-solubilized spheroplasts were subjected to immunoprecipitation using anti-Sec22p antibodies or preimmune IgG (preIgG). Cross-links were cleaved using β-mercaptoethanol in SDS sample buffer, and proteins cross-linked with Sec22p were analyzed by immunoblotting using the indicated antibodies. The asterisk indicates nonspecific bands. All experiments were performed multiple times with similar results, and the data shown are representative of all results.

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Sey1p physically interacts with ER SNAREs. (A and B) Sey1p physically interacts with the ER SNARE proteins Sec22p and Ufe1p. Microsomes isolated from BJ3505 were detergent-solubilized and incubated with anti-Sec22p antibodies (A), anti-Ufe1p antibodies (B), or preimmune IgG (control) in the presence of protein A Sepharose. Protein A Sepharose–bound material was then analyzed by immunoblotting using the indicated antibodies. (C) Sec18p regulates the interaction between Sec22p and Sey1p. BJ3505 microsomes were preincubated in the absence or presence of anti-Sec18p antibodies for 10 min at 4°C. After ATP/GTP was added, microsomes were further incubated for 40 min. During incubation, some samples received anti-Sec18p antibodies at the indicated time points. Sec22p was precipitated using anti-Sec22p antibody–conjugated Dynabeads, and bound proteins were analyzed by immunoblotting using the indicated antibodies. (D) Detection of an in vivo interaction between Sec22p and Sey1p by DSP cross-linking. BJ3505 spheroplasts were incubated in the absence or presence of 4 mM DSP for 30 min at 4°C. After DSP was quenched, detergent-solubilized spheroplasts were subjected to immunoprecipitation using anti-Sec22p antibodies or preimmune IgG (preIgG). Cross-links were cleaved using β-mercaptoethanol in SDS sample buffer, and proteins cross-linked with Sec22p were analyzed by immunoblotting using the indicated antibodies. The asterisk indicates nonspecific bands. All experiments were performed multiple times with similar results, and the data shown are representative of all results.

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Isolation, Incubation, In Vivo, Immunoprecipitation

    Tubulin-directed activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 P]GTP. GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

    doi:

    Figure Lengend Snippet: Tubulin-directed activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 P]GTP. GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.

    Article Snippet: The protein concentration of the extracts was adjusted to 1 mg/ml, and the extracts were incubated with GTP (1 mM)–bovine tubulin (10 μM) (Cytoskeleton, Denver, Colo.) in PEM buffer (pH 6.8).

    Techniques: Incubation, Concentration Assay, Activity Assay

    Modulation of nocodazole-stimulated tubulin GTPase activity by wt and mutated Op18. Tubulin (5 μM) was incubated at 37°C in the presence of [γ- 32 P]GTP in the absence (open bars) or presence (striped bars) of nocodazole (33 μM). The indicated Op18 derivative (16 μM) was also included in the reaction mixtures. The means of duplicate determinations of hydrolyzed GTP, after 120 min of incubation, are shown. Data are representative for at least two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

    doi:

    Figure Lengend Snippet: Modulation of nocodazole-stimulated tubulin GTPase activity by wt and mutated Op18. Tubulin (5 μM) was incubated at 37°C in the presence of [γ- 32 P]GTP in the absence (open bars) or presence (striped bars) of nocodazole (33 μM). The indicated Op18 derivative (16 μM) was also included in the reaction mixtures. The means of duplicate determinations of hydrolyzed GTP, after 120 min of incubation, are shown. Data are representative for at least two independent experiments.

    Article Snippet: The protein concentration of the extracts was adjusted to 1 mg/ml, and the extracts were incubated with GTP (1 mM)–bovine tubulin (10 μM) (Cytoskeleton, Denver, Colo.) in PEM buffer (pH 6.8).

    Techniques: Activity Assay, Incubation

    Activation of Rho GTPase coincides with OGG1-initiated BER. (A, B) Increased Rho–GTP levels in cultured cells exposed to oxidative stress. (A) MRC-5 and (B) MEF cells were exposed to GOx (100 ng/ml) and lysed at the times indicated. Rho–GTP levels were determined in 500 μg cell extracts by active Rho pull-down assays. (C) OGG1 depletion decreased Rho activation. MRC5 cells were transfected with siRNA to OGG1 or control siRNA and exposed to GO for 5 min. Rho–GTP was determined as for (A) and (B). (D, E) The OGG1-BER product 8-oxoG base increases Rho–GTP levels. (D) MRC5 and (E) MEF cells were exposed to 8-oxoG (10 μM) at 37 °C, cell extracts prepared at the indicated times, and Rho–GTP levels determined as for (A) and (B). (F) Lack of Rho activation in FapyG-exposed cells. Cells were exposed to FapyG (10 μM) and cell extracts were prepared at 5 min and Rho–GTP levels determined as for (A) and (B). As controls, calpeptin (a Rho activator) and 8-oxodG were used. (G) OGG1 depletion prevents Rho activation in 8-oxoG-exposed cells. MRC5 cells were transfected with OGG1 or control siRNA and exposed to 8-oxoG for 5 min. Rho–GTP was determined as for (A) and (B). Inset: extent of OGG1 downregulation by siRNA. (H) Lack of 8-oxoG-induced Rho activation in Ogg1 −/− MEFs. Cells ( Ogg1 −/− and Ogg1 +/+ ) were challenged with 8-oxoG, extracts were made at 5 min, and changes in Rho–GTP levels were determined by pull-down assays as for (A) and (B). (I) Activation of Rho GTPase in mouse lungs. Mice were challenged with 8-oxoG (1 μM) via the intranasal route and at 30 min active Rho was determined by pull-down assays. (J) Activation of Rho in lung extracts by 8-oxoG. Lung extracts from unchallenged mice were incubated with 10 μM 8-oxoG as indicated and changes in Rho–GTP levels determined as for (A) and (B). The graphs in (A), (B), (D), (E), and (J) depict the % of activated Rho. Band intensities were analyzed with ImageJ software and the percentage of active Rho in total Rho was calculated. (K) Expression of Rho isotypes in MRC-5 and Ogg1 −/− and Ogg1 +/+ MEF cells. Cell extracts (20 μg) were subjected to SDS–PAGE and RhoA, B, and C levels were determined by immunoblotting using type-specific Abs. GAPDH levels show equal loading. Cont, control. MEF, mouse embryonic fibroblasts; MRC5, human embryonic lung fibroblasts. Each experiment was repeated at least three times. * p

    Journal: Free radical biology & medicine

    Article Title: 8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerization

    doi: 10.1016/j.freeradbiomed.2014.03.030

    Figure Lengend Snippet: Activation of Rho GTPase coincides with OGG1-initiated BER. (A, B) Increased Rho–GTP levels in cultured cells exposed to oxidative stress. (A) MRC-5 and (B) MEF cells were exposed to GOx (100 ng/ml) and lysed at the times indicated. Rho–GTP levels were determined in 500 μg cell extracts by active Rho pull-down assays. (C) OGG1 depletion decreased Rho activation. MRC5 cells were transfected with siRNA to OGG1 or control siRNA and exposed to GO for 5 min. Rho–GTP was determined as for (A) and (B). (D, E) The OGG1-BER product 8-oxoG base increases Rho–GTP levels. (D) MRC5 and (E) MEF cells were exposed to 8-oxoG (10 μM) at 37 °C, cell extracts prepared at the indicated times, and Rho–GTP levels determined as for (A) and (B). (F) Lack of Rho activation in FapyG-exposed cells. Cells were exposed to FapyG (10 μM) and cell extracts were prepared at 5 min and Rho–GTP levels determined as for (A) and (B). As controls, calpeptin (a Rho activator) and 8-oxodG were used. (G) OGG1 depletion prevents Rho activation in 8-oxoG-exposed cells. MRC5 cells were transfected with OGG1 or control siRNA and exposed to 8-oxoG for 5 min. Rho–GTP was determined as for (A) and (B). Inset: extent of OGG1 downregulation by siRNA. (H) Lack of 8-oxoG-induced Rho activation in Ogg1 −/− MEFs. Cells ( Ogg1 −/− and Ogg1 +/+ ) were challenged with 8-oxoG, extracts were made at 5 min, and changes in Rho–GTP levels were determined by pull-down assays as for (A) and (B). (I) Activation of Rho GTPase in mouse lungs. Mice were challenged with 8-oxoG (1 μM) via the intranasal route and at 30 min active Rho was determined by pull-down assays. (J) Activation of Rho in lung extracts by 8-oxoG. Lung extracts from unchallenged mice were incubated with 10 μM 8-oxoG as indicated and changes in Rho–GTP levels determined as for (A) and (B). The graphs in (A), (B), (D), (E), and (J) depict the % of activated Rho. Band intensities were analyzed with ImageJ software and the percentage of active Rho in total Rho was calculated. (K) Expression of Rho isotypes in MRC-5 and Ogg1 −/− and Ogg1 +/+ MEF cells. Cell extracts (20 μg) were subjected to SDS–PAGE and RhoA, B, and C levels were determined by immunoblotting using type-specific Abs. GAPDH levels show equal loading. Cont, control. MEF, mouse embryonic fibroblasts; MRC5, human embryonic lung fibroblasts. Each experiment was repeated at least three times. * p

    Article Snippet: To determine the percentage of Rho–GTP, autoradiograms were quantified using ImageJ software.

    Techniques: Activation Assay, Cell Culture, Transfection, Mouse Assay, Incubation, Software, Expressing, SDS Page

    Effect of Archease on the single-turnover rate of RNA ligation by RtcB. ( A ) RNA ligation reactions with RtcB alone or with the inclusion of 100 nM Archease. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. ( B ) Plots of ligation product formation over time fitted to a single-exponential equation. Values are the mean ± SE for two separate experiments.

    Journal: Nucleic Acids Research

    Article Title: A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    doi: 10.1093/nar/gkt1375

    Figure Lengend Snippet: Effect of Archease on the single-turnover rate of RNA ligation by RtcB. ( A ) RNA ligation reactions with RtcB alone or with the inclusion of 100 nM Archease. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. ( B ) Plots of ligation product formation over time fitted to a single-exponential equation. Values are the mean ± SE for two separate experiments.

    Article Snippet: [14 C]GTP binding assays Binding assays were performed in 250 μl solutions consisting of 50 mM Bis–Tris buffer (pH 7.0), containing NaCl (300 mM), MnCl2 (2.0 mM) and [8-14 C]GTP (1.0 mM) (Moravek Biochemicals).

    Techniques: Ligation, Incubation

    Effect of Archease on the activity of active-site variants of RtcB in RNA ligation assays with GTP or ATP as a cofactor. ( A ) Reactions with GTP (0.10 mM) as a cofactor. ( B ) Reactions with ATP (0.10 mM) as a cofactor. Reaction mixtures included 100 nM Archease where indicated, and were incubated at 70°C for 30 min. ( C ) Graph of the ligation product obtained for each RtcB variant. Values are the mean ± SE for two separate experiments. ( D ) ATP-dependent K480A RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. ATP was included at 0.10 mM, and reaction mixtures were incubated at 70°C for 15 min. Values are the mean ± SE for three separate experiments. ( E ) Michaelis–Menten plot of reaction rate versus ATP cofactor concentration for K480A RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Values are the mean ± SE for three separate experiments. ( F ) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by K480A RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

    Journal: Nucleic Acids Research

    Article Title: A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    doi: 10.1093/nar/gkt1375

    Figure Lengend Snippet: Effect of Archease on the activity of active-site variants of RtcB in RNA ligation assays with GTP or ATP as a cofactor. ( A ) Reactions with GTP (0.10 mM) as a cofactor. ( B ) Reactions with ATP (0.10 mM) as a cofactor. Reaction mixtures included 100 nM Archease where indicated, and were incubated at 70°C for 30 min. ( C ) Graph of the ligation product obtained for each RtcB variant. Values are the mean ± SE for two separate experiments. ( D ) ATP-dependent K480A RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. ATP was included at 0.10 mM, and reaction mixtures were incubated at 70°C for 15 min. Values are the mean ± SE for three separate experiments. ( E ) Michaelis–Menten plot of reaction rate versus ATP cofactor concentration for K480A RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Values are the mean ± SE for three separate experiments. ( F ) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by K480A RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

    Article Snippet: [14 C]GTP binding assays Binding assays were performed in 250 μl solutions consisting of 50 mM Bis–Tris buffer (pH 7.0), containing NaCl (300 mM), MnCl2 (2.0 mM) and [8-14 C]GTP (1.0 mM) (Moravek Biochemicals).

    Techniques: Activity Assay, Ligation, Incubation, Variant Assay, Concentration Assay

    Structure-guided mutagenesis of conserved Archease residues. ( A , B ) Archease variants with alanine substitutions were tested for their ability to activate RtcB in RNA ligation reactions with GTP (A) or ATP (B) as a cofactor. Reaction mixtures included 100 nM Archease where specified and were incubated at 70°C for 30 min. ( C ) Graph of the ligation product obtained for each Archease variant. Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP or ATP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

    Journal: Nucleic Acids Research

    Article Title: A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    doi: 10.1093/nar/gkt1375

    Figure Lengend Snippet: Structure-guided mutagenesis of conserved Archease residues. ( A , B ) Archease variants with alanine substitutions were tested for their ability to activate RtcB in RNA ligation reactions with GTP (A) or ATP (B) as a cofactor. Reaction mixtures included 100 nM Archease where specified and were incubated at 70°C for 30 min. ( C ) Graph of the ligation product obtained for each Archease variant. Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP or ATP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

    Article Snippet: [14 C]GTP binding assays Binding assays were performed in 250 μl solutions consisting of 50 mM Bis–Tris buffer (pH 7.0), containing NaCl (300 mM), MnCl2 (2.0 mM) and [8-14 C]GTP (1.0 mM) (Moravek Biochemicals).

    Techniques: Mutagenesis, Ligation, Incubation, Variant Assay

    RNA ligation reactions demonstrating the NTP cofactor specificity of RtcB and an active-site view of the RtcB–pG intermediate. ( A ) Reactions testing NTP cofactor specificity with RtcB alone or RtcB with 100 nM Archease. NTP cofactors were tested at 0.10 mM, and reaction mixtures were incubated at 70°C for 30 min. ( B ) Graph of the ligation product obtained for each NTP cofactor. Values are the mean ± SE for two separate experiments. ( C ) ATP-dependent RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. Reaction mixtures were incubated at 70°C for 20 min. Values are the mean ± SE for three separate experiments. ( D ) Crystal structure of the P. horikoshii RtcB–pG intermediate (PDB entry 4it0) illustrating the residues that contact the guanine nucleobase. ( E–I ) Michaelis–Menten plots of reaction rate versus NTP cofactor concentration for RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Where indicated, Archease was included at a concentration of 100 nM for reactions with GTP, dGTP and ITP, while reactions with ATP included 800 nM Archease. Values are the mean ± SE for three separate experiments. ( J ) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

    Journal: Nucleic Acids Research

    Article Title: A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    doi: 10.1093/nar/gkt1375

    Figure Lengend Snippet: RNA ligation reactions demonstrating the NTP cofactor specificity of RtcB and an active-site view of the RtcB–pG intermediate. ( A ) Reactions testing NTP cofactor specificity with RtcB alone or RtcB with 100 nM Archease. NTP cofactors were tested at 0.10 mM, and reaction mixtures were incubated at 70°C for 30 min. ( B ) Graph of the ligation product obtained for each NTP cofactor. Values are the mean ± SE for two separate experiments. ( C ) ATP-dependent RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. Reaction mixtures were incubated at 70°C for 20 min. Values are the mean ± SE for three separate experiments. ( D ) Crystal structure of the P. horikoshii RtcB–pG intermediate (PDB entry 4it0) illustrating the residues that contact the guanine nucleobase. ( E–I ) Michaelis–Menten plots of reaction rate versus NTP cofactor concentration for RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Where indicated, Archease was included at a concentration of 100 nM for reactions with GTP, dGTP and ITP, while reactions with ATP included 800 nM Archease. Values are the mean ± SE for three separate experiments. ( J ) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

    Article Snippet: [14 C]GTP binding assays Binding assays were performed in 250 μl solutions consisting of 50 mM Bis–Tris buffer (pH 7.0), containing NaCl (300 mM), MnCl2 (2.0 mM) and [8-14 C]GTP (1.0 mM) (Moravek Biochemicals).

    Techniques: Ligation, Incubation, Concentration Assay

    The three nucleotidyl transfer steps of catalysis by RtcB, a putative tRNA splicing operon and the titration of Archease into RNA ligation reactions with RtcB. ( A ) The three nucleotidyl transfer steps of catalysis by RtcB are (1) RtcB guanylylation, (2) RNA 3′-P guanylylation and (3) phosphodiester bond formation. ( B ) The operon organization of rtcB and archease in diverse bacteria ( Pelobacter propionicus , Synechococcus sp. JA-3-3Ab and Syntrophus aciditrophicus ) and archaea ( Halobacterium sp. NRC-1 , Methanosaeta thermophila , P. horikoshii and Thermococcus kodakarensis ). ( C ) RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). (RNA substrates are shown at top.) Reaction mixtures were incubated at 70°C for 30 min, and quenched with an equal volume of RNA gel-loading buffer. The reaction products were resolved by electrophoresis through an 18% w/v urea–polyacrylamide gel and visualized by fluorescence scanning of the FAM label. ( D ) Ligation product (nM) plotted versus Archease concentration (nM). Values are the mean ± SE for three separate experiments.

    Journal: Nucleic Acids Research

    Article Title: A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    doi: 10.1093/nar/gkt1375

    Figure Lengend Snippet: The three nucleotidyl transfer steps of catalysis by RtcB, a putative tRNA splicing operon and the titration of Archease into RNA ligation reactions with RtcB. ( A ) The three nucleotidyl transfer steps of catalysis by RtcB are (1) RtcB guanylylation, (2) RNA 3′-P guanylylation and (3) phosphodiester bond formation. ( B ) The operon organization of rtcB and archease in diverse bacteria ( Pelobacter propionicus , Synechococcus sp. JA-3-3Ab and Syntrophus aciditrophicus ) and archaea ( Halobacterium sp. NRC-1 , Methanosaeta thermophila , P. horikoshii and Thermococcus kodakarensis ). ( C ) RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). (RNA substrates are shown at top.) Reaction mixtures were incubated at 70°C for 30 min, and quenched with an equal volume of RNA gel-loading buffer. The reaction products were resolved by electrophoresis through an 18% w/v urea–polyacrylamide gel and visualized by fluorescence scanning of the FAM label. ( D ) Ligation product (nM) plotted versus Archease concentration (nM). Values are the mean ± SE for three separate experiments.

    Article Snippet: [14 C]GTP binding assays Binding assays were performed in 250 μl solutions consisting of 50 mM Bis–Tris buffer (pH 7.0), containing NaCl (300 mM), MnCl2 (2.0 mM) and [8-14 C]GTP (1.0 mM) (Moravek Biochemicals).

    Techniques: Titration, Ligation, Incubation, Electrophoresis, Fluorescence, Concentration Assay

    Effects of Archease on the rate of RtcB-catalyzed guanylylation of RNA with a 2′-F/3′-P terminus. RtcB was pre-guanylylated by incubation with GTP and Mn(II), and the 3′ RNA fragment was not included to prevent ligation. ( A ) The guanylylation rate of a 2′-F/3′-P RNA terminus by RtcB alone or with the inclusion of 100 nM Archease. RNA guanylylation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM) and RNA substrate (1.0 μM). (RNA substrate is shown at top.) Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. ( B ) Plots of RNA–ppG product formation over time fitted to a single-exponential equation. ( C ) Plots of RtcB-catalyzed RNA–ppG product formation over time in reactions that had the 2′-F/3′-P RNA substrate in excess (0.5 μM RtcB and 1.0 μM RNA substrate). Archease (100 nM) was added where indicated. Values in the plots are the mean ± SE for two separate experiments.

    Journal: Nucleic Acids Research

    Article Title: A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    doi: 10.1093/nar/gkt1375

    Figure Lengend Snippet: Effects of Archease on the rate of RtcB-catalyzed guanylylation of RNA with a 2′-F/3′-P terminus. RtcB was pre-guanylylated by incubation with GTP and Mn(II), and the 3′ RNA fragment was not included to prevent ligation. ( A ) The guanylylation rate of a 2′-F/3′-P RNA terminus by RtcB alone or with the inclusion of 100 nM Archease. RNA guanylylation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl 2 (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM) and RNA substrate (1.0 μM). (RNA substrate is shown at top.) Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. ( B ) Plots of RNA–ppG product formation over time fitted to a single-exponential equation. ( C ) Plots of RtcB-catalyzed RNA–ppG product formation over time in reactions that had the 2′-F/3′-P RNA substrate in excess (0.5 μM RtcB and 1.0 μM RNA substrate). Archease (100 nM) was added where indicated. Values in the plots are the mean ± SE for two separate experiments.

    Article Snippet: [14 C]GTP binding assays Binding assays were performed in 250 μl solutions consisting of 50 mM Bis–Tris buffer (pH 7.0), containing NaCl (300 mM), MnCl2 (2.0 mM) and [8-14 C]GTP (1.0 mM) (Moravek Biochemicals).

    Techniques: Incubation, Ligation

    Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than GTP/GDP interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: Close-up stereo views of the switch I and II regions in EF-Tu ( a ) and MM1309 ( b ). The bound GMPPNP molecule and the Mg 2+ ion, and the EF-Tu and MM1309 residues in the switch I and II regions, which are involved in the GMPPNP interactions, are shown as stick models. The EF-Tu and MM1309 residues that are involved in the domain–domain interactions are also shown as stick models. The switch I and II regions of MM1309 are involved in domain–domain interactions, rather than GTP/GDP interactions. The switch I and II regions are colored pink and green , respectively. Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques:

    ITC analysis. The upper and lower panels display the ITC titration curves and the binding isotherms, respectively, for MM1309 with GTP·Mg 2+ ( a ), GTP without Mg 2+ ( b ), GDP·Mg 2+ ( c ), and GMPPNP·Mg 2+ ( d ). N , the binding stoichiometry; K b , the observed binding constant; K d ( K d = 1/ K b ), the dissociation constant; ∆ H , the binding enthalpy; ∆ S , the binding entropy

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: ITC analysis. The upper and lower panels display the ITC titration curves and the binding isotherms, respectively, for MM1309 with GTP·Mg 2+ ( a ), GTP without Mg 2+ ( b ), GDP·Mg 2+ ( c ), and GMPPNP·Mg 2+ ( d ). N , the binding stoichiometry; K b , the observed binding constant; K d ( K d = 1/ K b ), the dissociation constant; ∆ H , the binding enthalpy; ∆ S , the binding entropy

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques: Titration, Binding Assay

    Stereo views of the GTP binding sites. a The bound GMPPNP molecule in the T. aquaticus EF-Tu·GMPPNP·Mg 2+ structure. b The bound GMPPNP molecule in the MM1309·GMPPNP·Mg 2+ structure. The F o – F c omit map (contoured at 3.3 σ) of the bound GMPPNP·Mg 2+ in the MM1309 active site. c , d Close-up stereo views around the γ-phosphate group of the bound GMPPNP in T. aquaticus EF-Tu·GMPPNP·Mg 2+ ( c ) and MM1309·GMPPNP·Mg 2+ ( d ). The amino acid residues surrounding the phosphate groups and the magnesium ions of the bound GMPPNP·Mg 2+ are depicted by stick models. e The bound GDP molecule in the MM1309·GDP structure. The F o – F c omit map (contoured at 4.0 σ) of the bound GDP·Mg 2+ in the MM1309 active site. f The GTP binding site in the MM1309 apo form. The MM1309 residues that are located close to the bound guanine nucleotide are represented as stick models. The P-loop motifs (Gly17–Thr25 in EF-Tu and Gly7–Thr15 in MM1309) are shown in sky blue . The switch I regions are colored pink . Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: Stereo views of the GTP binding sites. a The bound GMPPNP molecule in the T. aquaticus EF-Tu·GMPPNP·Mg 2+ structure. b The bound GMPPNP molecule in the MM1309·GMPPNP·Mg 2+ structure. The F o – F c omit map (contoured at 3.3 σ) of the bound GMPPNP·Mg 2+ in the MM1309 active site. c , d Close-up stereo views around the γ-phosphate group of the bound GMPPNP in T. aquaticus EF-Tu·GMPPNP·Mg 2+ ( c ) and MM1309·GMPPNP·Mg 2+ ( d ). The amino acid residues surrounding the phosphate groups and the magnesium ions of the bound GMPPNP·Mg 2+ are depicted by stick models. e The bound GDP molecule in the MM1309·GDP structure. The F o – F c omit map (contoured at 4.0 σ) of the bound GDP·Mg 2+ in the MM1309 active site. f The GTP binding site in the MM1309 apo form. The MM1309 residues that are located close to the bound guanine nucleotide are represented as stick models. The P-loop motifs (Gly17–Thr25 in EF-Tu and Gly7–Thr15 in MM1309) are shown in sky blue . The switch I regions are colored pink . Transparent ribbon models of EF-Tu ( blue ) and MM1309 ( white ) are visible in the background

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques: Binding Assay

    Superposition of MM1309 with EF-Tu, aSelB, and aIF2γ, represented by ribbon models. a Superposition of the MM1309 structures in the GMPPNP-bound, GDP-bound, and apo forms. b Superposition of MM1309 with T. aquaticus EF-Tu in the GTP-bound form (PDB code: 1TTT). c Superposition of MM1309 with T. aquaticus EF-Tu in the GDP-bound form (PDB code: 1TUI). d Superposition of MM1309 with M. maripaludis aSelB (PDB code: 4ACA) and with P. abyssi aIF2γ (PDB code: 1KK0)

    Journal: Journal of Structural and Functional Genomics

    Article Title: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

    doi: 10.1007/s10969-015-9193-6

    Figure Lengend Snippet: Superposition of MM1309 with EF-Tu, aSelB, and aIF2γ, represented by ribbon models. a Superposition of the MM1309 structures in the GMPPNP-bound, GDP-bound, and apo forms. b Superposition of MM1309 with T. aquaticus EF-Tu in the GTP-bound form (PDB code: 1TTT). c Superposition of MM1309 with T. aquaticus EF-Tu in the GDP-bound form (PDB code: 1TUI). d Superposition of MM1309 with M. maripaludis aSelB (PDB code: 4ACA) and with P. abyssi aIF2γ (PDB code: 1KK0)

    Article Snippet: [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -triphosphate] (Mant-GTP) and [2′-/3′-O -(N -methylanthraniloyl)guanosine-5′-O -diphosphate] (Mant-GDP) were purchased from Jena Bioscience (Germany).

    Techniques:

    Phosphorylation of the XJ TGB1 protein in vitro and in vivo . (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJ TGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJ TGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ- 32 P]ATP or [γ- 32 P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJ TGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJ TGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJ TGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJ RNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJ TGB1 protein. (D) In vivo phosphorylation of XJ TGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C . (This figure is available in colour at JXB online.)

    Journal: Journal of Experimental Botany

    Article Title: Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots

    doi: 10.1093/jxb/erv237

    Figure Lengend Snippet: Phosphorylation of the XJ TGB1 protein in vitro and in vivo . (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJ TGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJ TGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ- 32 P]ATP or [γ- 32 P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJ TGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJ TGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJ TGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJ RNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJ TGB1 protein. (D) In vivo phosphorylation of XJ TGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C . (This figure is available in colour at JXB online.)

    Article Snippet: Assays were performed with 1 μg of N. benthamiana soluble protein extracts, or 0.1 μg of recombinant CK2α, and 1 μg of purified XJ TGB1 protein or its mutants in a final volume of 10 μl of 25mM Tris/HCl (pH 7.4), 10mM MgCl2 , and 1 μl [γ-32 P]ATP or GTP (10 μCi, ~3000 Ci mmol–1 ; Perkin Elmer).

    Techniques: In Vitro, In Vivo, Staining, Recombinant, Purification, Molecular Weight, SDS Page, Radioactivity, Autoradiography, Western Blot, Negative Control, Immunoprecipitation

    In vitro phosphorylation of XJ TGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of XJ TGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of XJ TGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B . (F) Co-localization of the GFP: XJ TGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP: XJ TGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)

    Journal: Journal of Experimental Botany

    Article Title: Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots

    doi: 10.1093/jxb/erv237

    Figure Lengend Snippet: In vitro phosphorylation of XJ TGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of XJ TGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of XJ TGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B . (F) Co-localization of the GFP: XJ TGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP: XJ TGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)

    Article Snippet: Assays were performed with 1 μg of N. benthamiana soluble protein extracts, or 0.1 μg of recombinant CK2α, and 1 μg of purified XJ TGB1 protein or its mutants in a final volume of 10 μl of 25mM Tris/HCl (pH 7.4), 10mM MgCl2 , and 1 μl [γ-32 P]ATP or GTP (10 μCi, ~3000 Ci mmol–1 ; Perkin Elmer).

    Techniques: In Vitro, Recombinant, SDS Page, Purification, Staining

    Bleomycin decreased vascular endothelial growth factor receptor 2 (VEGFR2) and guanosine triphosphate cyclohydrolase-1 (GTPCH-1) levels in WT mice. VEGFR2, endothelial nitric oxide synthase (eNOS) and GTPCH-1 protein content in the lung were evaluated by western blot analysis in WT and R 213 G mice at 22 days of age after exposure to PBS (10 μL for 9 doses) or Bleomycin (3 units/kg/dose dissolved in 10 μL of PBS for 9 doses). 25 μg lung protein was loaded onto the gels. Representative blots are shown along with optical density normalized to β-actin and expressed relative to WT control mice. ( a ) VEGFR2 ( b ) eNOS ( c ) Representative blots for VEGFR2 and eNOS ( d ) GTPCH-1, ( e ) Representative blot for GTPCH-1 * p

    Journal: Antioxidants

    Article Title: Redistribution of Extracellular Superoxide Dismutase Causes Neonatal Pulmonary Vascular Remodeling and PH but Protects Against Experimental Bronchopulmonary Dysplasia

    doi: 10.3390/antiox7030042

    Figure Lengend Snippet: Bleomycin decreased vascular endothelial growth factor receptor 2 (VEGFR2) and guanosine triphosphate cyclohydrolase-1 (GTPCH-1) levels in WT mice. VEGFR2, endothelial nitric oxide synthase (eNOS) and GTPCH-1 protein content in the lung were evaluated by western blot analysis in WT and R 213 G mice at 22 days of age after exposure to PBS (10 μL for 9 doses) or Bleomycin (3 units/kg/dose dissolved in 10 μL of PBS for 9 doses). 25 μg lung protein was loaded onto the gels. Representative blots are shown along with optical density normalized to β-actin and expressed relative to WT control mice. ( a ) VEGFR2 ( b ) eNOS ( c ) Representative blots for VEGFR2 and eNOS ( d ) GTPCH-1, ( e ) Representative blot for GTPCH-1 * p

    Article Snippet: Blots were blocked for 1 h using 5% nonfat milk in 1× TBST, then probed overnight at 4 °C using the following antibodies: catalase (1:500, Abcam, Cambridge, UK), superoxide dismutase 1 (SOD1) (1:1000, Abcam, Cambridge, UK), superoxide dismutase 2 (SOD2) (1:1000, Millipore, Billerica, MA, USA), SOD3 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), vascular endothelial growth factor receptor 2 (VEGFR2) (1:500, Cell Signaling, Danvers, MA, USA), endothelial nitric oxide synthase (eNOS) (1:1000, BD Biosciences, San Jose, CA, USA), guanosine triphosphate cyclohydrolase-1 (GTPCH-1) (1:1000, Abcam, Cambridge, UK).

    Techniques: Mouse Assay, Western Blot